The human brain is arguably the most complex biological structure. Understanding how many different cell types exist in the human brain and mapping neural connections are critical tasks to better understand the development and function of the brain. This is particularly challenging in the human brain due to inherent limitations of working with postmortem tissue. This grant is specifically addressing these tasks in the human brain as well as a closely related non-human primate, Rhesus macaque, and a commonly studied mammalian organism, the mouse. The objective of this proposal is to employ novel methods and approaches to generate a systematic inventory/census of cell types and connections in the developing and adult human, macaque monkey and mouse prefrontal cortex (PFC). We have chosen PFC for this project due both to its importance in higher cognitive functions as well as for the alterations observed in PFC in certain psychiatric and neurological disorders. To generate a census of component cells in the PFC, we will use single cell RNA-seq to profile the transcriptomes of single cells isolated in a cell-type specific way through the use of viral-mediated tagging of ribosomes and axonal tracing methods. To apply these advanced techniques to post-mortem human brain, we will implement a novel tissue processing protocol [Hibernation-Cryopreservation combined with a Pulsatile Perfusion Hibernation System] to keep post-mortem brains in prolonged hibernation. This allows for the stabilization of nucleic acids, the concurrent collection of live single cells, and the applicationof classical and advanced methods for the identification of neuronal pathways. We will also develop agnostic and integrative computational methods to create a taxonomy of cell types based on molecular identity and connectivity. These features will be compared across species, ages, and sexes. There are four major distinguishing aspects of this application: (1) implementation of novel approaches developed to extend tissue integrity and viability of the postmortem human brain such that we can (2) perform single cell RNA-seq, (3) trace connections and examine cell morphology in the postmortem human brain, which is not amenable to classical experimentations~ and (4) develop novel analytical tools and approaches. This pilot project and methodologies directly address the goals of this BRAIN Initiative RFA and are designed to demonstrate their utility and scalability to ultimately complete a comprehensive cell census of the entire human brain in healthy and disease states.